Short-term carcinogenicity study of N-methyl-N-nitrosourea in FVB-Trp53 heterozygous mice

Carcinogenicity tests predict the tumorigenic potential of various substances in the human body by studying tumor induction in experimental animals. There is a need for studies that explore the use of FVB/N-Trp53em2Hwl/Korl (FVB-Trp53+/-) mice, created by TALEN-mediated gene targeting in Korea, in carcinogenicity tests. This study was performed to determine whether FVB-Trp53+/- mice are a suitable model for short-term carcinogenicity studies. To compare the carcinogenicity at different concentrations, 25, 50, and 75 mg/kg of N-methyl-N-nitrosourea (MNU), a known carcinogen, were administered intraperitoneally to FVB-Trp53+/- and wild-type male mice. After 26 weeks, the survival rate was significantly reduced in FVB-Trp53+/- mice compared to the wild-type mice in the 50 and 75 mg/kg groups. The incidence of thymic malignant lymphoma (TML) in the 50 and 75 mg/kg groups was 54.2 and 59.1% in FVB-Trp53+/- male mice, respectively. TML metastasized to the lungs, spleen, lymph nodes, liver, kidney, and heart in FVB-Trp53+/- male mice. Furthermore, the incidence of primary lung tumors, such as adenomas and adenocarcinomas, was 65.4, 62.5, and 45.4% in the FVB-Trp53+/- mice of the 25, 50, and 75 mg/kg groups, respectively. The main tumor types in FVB-Trp53+/- mice were TML and primary lung tumors, regardless of the dose of MNU administered. These results suggest that systemic tumors may result from malfunctions in the p53 gene and pathway, which is an important factor in the pathogenesis of human cancers. Therefore, FVB-Trp53 heterozygous mice are suitable for short-term carcinogenicity tests using positive carcinogens, and that the best result using MNU, a positive carcinogen, might have a single dose of 50 mg/kg.


Introduction
Carcinogenicity tests use experimental animals to predict the risk of tumorigenesis from exposure to various substances [1]. These tests can be performed for drugs suspected of having a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 bedding. The facility was maintained in an air-conditioned system at 22 ± 2˚C, at a relative humidity of 50 ± 10%, and a 12 h light /12 h dark cycle. To diminish distress, pulp house and wood chew block were provided for mice. The mice were weighed once a week to check for weight changes by trained researchers. The activity, appearance, and survival of the mice were observed once daily. Humane euthanasia performed on the day reaching 25% of activity score. Mice found dead were necropsied immediately. All animal experiments were approved by the Institutional Animal Care and Use Committee of Konkuk University, Korea (KU20081).

Histopathological analysis
Except for unscheduled death, mice were sacrificed 26 weeks after MNU administration. The lungs, spleen, and liver were separated and weighed, and relative organ weights (organ weight to body weight ratio) were calculated. All organs collected for histopathological evaluation were fixed by embedding in 10% neutral-buffered formalin and processed to prepare paraffin blocks. The prepared paraffin blocks were cut into 4 μm sections and attached to slides. After deparaffinization, the slides were stained with hematoxylin and eosin. The prepared slides were observed under a BX51 microscope (Olympus, Japan) and analyzed using the DP71 (Olympus) program.

Immunohistochemistry
Paraffin blocks were sliced to a thickness of 4 μm and attached to a silane-coated slide (Muto Pure Chemicals Co., Ltd., Japan). Each procedure was performed according to the ABC kit protocol (Vector Laboratories, USA). After deparaffinization and rehydration, slides were boiled in 0.1 M sodium citrate buffer (pH 6.0) in a microwave oven for antigen retrieval. After cooling the slides to room temperature, 3% H 2 O 2 in methanol was used to block the endogenous peroxidase activity. To suppress non-specific reactions, blocking serum (Vector Laboratories) was applied to the tissue. PCNA antibody (Abcam, ab92552, diluted 1:200), prosurfactant protein C (SPC, Abcam, ab90716, diluted 1:1000), and ubiquitin antibody (CC10, Abcam, ab213203, diluted 1:4000) were used as the primary antibodies. Biotinylated antibody (Vector Laboratories, USA) was used as a secondary antibody. It was then detected using the DAB Peroxidase Substrate Kit (Vector Laboratories, USA). All the slides were counterstained with hematoxylin (Gill III hematoxylin, Thermo, USA).

Body weight changes
The weights of mice in each group were measured once per week. The body weight of FVB-p53 +/and wild-type mice gradually increased over time in the 0 (untreated control), 25, 50, and 75 mg/kg groups. There was no significant difference in body weight between heterozygous and wild-type mice in the same MNU administration group (Fig 1A, 1C, 1E and 1G).

Mouse survival rate
At the end of the study at 26 weeks, the survival rate in the 25 mg/kg group was 84.6% (22 of 26 mice) in FVB-Trp53 +/mice and 100% (19 of 19 mice) in wild-type mice ( Fig 1D). The survival rate in the 50 mg/kg group was 62.5% (15 of 24 mice) in FVB-Trp53 +/mice and 96.2% (25 of 26 mice) in wild-type mice ( Fig 1F). The survival rate in the 75 mg/kg group was 31.8% (7 of 22 mice) in FVB-Trp53 +/mice and 90% (18 of 20 mice) in wild-type mice ( Fig 1H). The survival rates of 50 and 75 mg/kg groups in FVB-p53 +/mice was significantly lower than those of wild-type mice (Fig 1F and 1H).

Hematology and serum chemistry
Thirty hematology tests and 19 serum chemistry tests were performed. Hematological analysis revealed that the percentage of neutrophils in the 25 mg/kg group was significantly higher in FVB-Trp53 +/than in wild-type mice. In the 50 mg/kg group, the number of RBCs and neutrophils and the percentage of neutrophils were significantly higher in FVB-Trp53 +/than in wild-type mice; however, MCH and the percentage of lymphocytes were significantly lower in FVB-Trp53 +/than in wild-type mice. In the 75 mg/kg group, MCV, MPV, and PDW were significantly lower in FVB-Trp53 +/than in wild-type mice (S1 Table). Some hematology tests exhibited a significant difference between the groups; however, all results were within the reference range.
The results of the serum chemistry tests in the 25 mg/kg group show that IP significantly increased and triglyceride significantly decreased in FVB-Trp53 +/mice compared to wildtype mice. In the 75 mg/kg group, glucose and IP were significantly higher in FVB-Trp53 +/mice than in wild-type mice; however, calcium levels were significantly lower in FVB-Trp53 +/mice than in wild-type mice (S2 Table). Similar to the hematological analysis, some serum chemistry tests exhibited a significant difference between groups; however, all results were within the reference range.

Organ to body weight ratio
Organ weights were measured in all surviving mice until 26 weeks after MNU administration. In the 25 and 75 mg/kg groups, no significant difference was observed between the lung, liver, and spleen weights (%) of FVB-Trp53 +/and wild-type mice. In the 50 mg/kg group, the relative lung and spleen weights of FVB-Trp53 +/mice were significantly higher than those of wild-type mice (Fig 2).

Gross lesion
During the experiment, all moribund mice were sacrificed, and all surviving mice were necropsied 26 weeks after MNU administration. In FVB-Trp53 +/mice, lung nodules were found in 76.9% of the 25 mg/kg group, 91.7% of the 50 mg/kg group, and 90.9% of the 75 mg/kg group (Fig 3A-3C). However, in wild type mice, lung nodules were found in 52.6%, 76.9% and 90% of the 25, 50, and 75 mg/kg groups, respectively.
In the 25 mg/kg group, enlarged thymus ( Fig 3D) and splenomegaly ( Fig 3G) were found in 11.5% and 7.7% of FVB-Trp53 +/mice, respectively, while none were found in wild-type mice. In the 50 mg/kg group, enlarged thymus ( Fig 3E) and splenomegaly ( Fig 3H) were found in 54.2% and 29.2% of FVB-Trp53 +/mice, respectively, while none were found in wild-type mice. In the 75 mg/kg group, enlarged thymus ( Fig 3F) and splenomegaly ( Fig 3I) were found in 59.1% and 27.3% of FVB-Trp53 +/mice, respectively, and in only one wild-type mouse. Subcutaneous masses were observed in one FVB-Trp53 +/mouse of the 25 mg/kg group ( Fig  3J) and in three FVB-Trp53 +/of the 75 mg/kg group (Fig 3K).

Histopathology
Based on tumor cell morphology observed during histopathological analysis of the lung nodules, primary lung tumors were divided into adenoma and adenocarcinoma (Fig 4A, 4C and  4E). In addition, histopathological analysis of enlarged thymus tissues revealed thymic malignant lymphoma (TML) as the primary tumor (Fig 4B, 4D and 4F). Metastatic TMLs were found in multiple organs, such as the lungs, liver, enlarged spleens, kidneys, lymph nodes, and heart (Table 1). TML metastasis in the lungs was mainly observed in the peribronchiolar or perivascular regions. Interestingly, two types of malignant tumors, primary lung adenocarcinoma and TML lung metastasis, were observed in the lungs of several mice, respectively ( Table 1). The subcutaneous masses were identified as malignant fibrosarcomas.
The overall tumor incidence (Table 1) in FVB-Trp53 +/mice was 80.8% and 100% in the 25 and 50 mg/kg groups, respectively, which was significantly higher than the 52.6% and 76.9% in wild-type mice (Fig 5A). Lung tumor incidence involving adenoma, adenocarcinoma, and TML lung metastasis in FVB-Trp53 +/mice were 76.9%, 91.7%, and 90.9% in the 25, 50, and 75 mg/kg groups, respectively, which were higher than that of wild-type mice, but the difference was not significant (Fig 5B). The incidence rates of malignant tumors in the lungs of FVB-Trp53 +/mice were 42.3%, 62.5%, and 63.7% in the 25, 50, and 75 mg/kg groups, respectively, which were significantly higher than the 10.5%, 11.5%, and 25.0% in wild-type mice ( Fig 5C). The incidence rates of TML in the thymus of FVB-Trp53 +/mice were 54.2% and 59.1% in the 50 mg/kg and 75 mg/kg groups, respectively, which were significantly higher than the 0% and 5% in wild-type mice (Fig 5D). Metastasis of TML was observed in 29.2% and 45.5% of FVB-Trp53 +/mice in the 50 and 75 mg/kg groups, respectively, which was significantly higher than that in wild-type mice (0% and 5%, respectively) ( Fig 5E).

Immunohistochemistry
Immunohistochemical staining was performed to confirm the characteristics of the lung tumors. Proliferating cell nuclear antigen (PCNA), prosurfactant protein C (SPC), and CC10 (club cell 10-kDa protein) were used. PCNA is a cell proliferation marker that is stained in adenomas and adenocarcinomas of the lungs, TML, and TML lung metastases. In particular, malignant tumors, such as adenocarcinoma, TML, and TML lung metastases, were more strongly stained with PCNA (Fig 6B, 6E and 6F). In lung adenoma, PCNA was only expressed in some cells (Fig 6A). SPC, known as an alveolar type 2 cell marker, was observed in adenomas and adenocarcinomas in the lungs (Fig 6C and 6D). SPC staining was more strongly observed in adenocarcinomas than adenomas. However, SPC was not expressed in TML and TLM lung metastases. CC10, a Clara cell marker, was not expressed in adenoma and adenocarcinoma (Fig 6G) in the lungs and TML, but CC10 expression was observed in the bronchial epithelium at the center of TML lung metastasis (Fig 6H).
A higher incidence (> 40%) of malignant lung tumors was observed in the 25-75 mg/kg group in this study than in previous studies [17,27]. Interestingly, lung adenocarcinoma was observed in 30.8, 33.3, and 18.2% of FVB-Trp53 +/mice in the 25, 50, and 75 mg/kg groups, respectively. Furthermore, the incidence of primary lung tumors, such as adenoma and adenocarcinoma, which has not been reported in previous papers [17,27], was 65.4, 62.5, and 45.4% in the 25, 50, and 75 mg/kg groups of FVB-Trp53 +/mice, respectively, supporting the reports that FVB/N mice are susceptible to lung tumorigenesis [28,29]. The main tumor types in FVB-Trp53 +/mice were TML and primary lung tumors, regardless of the dose of MNU administered. However, the main tumor type in C57BL/6 background Trp53 +/mice is malignant lymphoma in the thymus and spleen [17,27,30,31].
Based on our data and a previously published paper, MNU could be used as a positive control for FVB-Trp53 +/mice in short-term carcinogenicity studies. We found that both the 50 and 75 mg/kg groups of FVB-Trp53 +/mice had similar incidences of TML and lung tumors. Furthermore, the survival rate in the 50 mg/kg group was higher than that in the 75 mg/kg group. Therefore, 50 mg/kg in FVB-Trp53 +/mice was considered a suitable concentration for positive control in a short-term carcinogenic study.
Immunohistochemical staining was performed to investigate the pattern of primary lung tumors in FVB-Trp53 mice. PCNA is considered a key prognostic index for cancer [32]. PCNA in malignant tumors of the lungs was more prominently stained than in lung adenoma. CC10 is an anti-inflammatory protein produced by epithelial cells in the lungs, rarely found in human non-small cell carcinoma or tumor cell lines, and abundantly produced in progenitor cells of normal and neoplastic epithelium [33]. CC10 expression was observed only in the bronchiolar epithelium at the center of TML lung metastasis, while low CC10 expression was observed in primary adenoma or adenocarcinoma of the lungs. Nonetheless, it is difficult to conclude that anti-inflammatory response is increased in TML lung metastasis. SPC is a pulmonary surfactant protein C that is one of the four surfactant proteins produced by type II alveolar epithelial cells [34]. The expression of SPC decreases significantly in various types of lung injury and is associated with ACE apoptosis [35]. The primary adenoma and adenocarcinoma in the lungs in this study showed SPC expression, which indicates the possibility of a type II alveolar cell-derived primary lung tumor.
In summary, FVB-Trp53 heterozygous mice were used for short-term carcinogenicity tests, using MNU as a positive control. A single dose of 50 mg/kg MNU in FVB-Trp53 +/might be a suitable concentration for positive control in short-term carcinogenic studies.
Supporting information S1